Inhibition of tumorigenicity and metastasis of human bladder cancer growing in athymic mice by interferon-beta gene therapy results partially from various antiangiogenic effects including endothelial cell apoptosis.

We determined whether the IFN-beta gene could suppress angiogenesis, tumor growth, and metastasis of human bladder transitional cell carcinoma. The highly tumorigenic and metastatic 253J B-V(R) human bladder transitional cell carcinoma (TCC) cell line (resistant to the antiproliferative effects of IFN-beta) was infected in vitro with adenoviral beta-galactosidase (Ad-LacZ), murine adenoviral IFN-beta (Ad-mIFN-beta), or human adenoviral IFN-beta (Ad-hIFN-beta) and implanted into the bladders of athymic nude mice. Ad-mIFN-beta and Ad-hIFN-beta were used because of the species specificity of IFN-beta. The transient production of mIFN-beta and hIFN-beta from the infected 253JB-V(R) tumor cells significantly inhibited tumorigenicity and spontaneous lymph node metastasis. Subsequently, the 253J B-V(R) cells were implanted into the subcutis of athymic nude mice, and established tumors were treated by direct intratumoral injection with Ad-mIFN-beta, Ad-hIFN-beta, Ad-LacZ, or PBS. By in situ hybridization (ISH) and immunohistochemical analysis (IHC), expression of hIFN-beta and mIFN-beta mRNA and protein within the tumors was demonstrated after Ad-hIFN-beta and Ad-mIFN-beta gene therapy, respectively. The therapy also induced necrosis in both the Ad-mIFN-beta- and Ad-hIFN-beta-treated tumors. IHC revealed decreased tumor cell proliferation and the sequestration of activated macrophages within the tumors after Ad-mIFN-beta therapy. In addition, the expression of the proangiogenic factors bFGF, and MMP-9 protein (by IHC) was significantly down-regulated by Ad-hIFN-beta gene therapy. Double-immunofluorescent IHC for terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) and CD-31 demonstrated tumor and endothelial cell apoptosis in those tumors treated with Ad-hIFN-beta gene therapy. Tumor-induced angiogenesis, as determined by the microvessel density, was decreased in tumors treated with both Ad-mIFN-beta and Ad-hIFN-beta. These data suggest that the inhibition of tumorigenicity and the metastasis of the 253J B-V(R) cells after infection with Ad-IFN-beta is caused by the inhibition of angiogenesis and the activation of host effector cells.     

The effect of heat-treated human bone morphogenetic protein on clinical implantation

For the clinical usage of human-derived bones, it is necessary to treat bones to reduce the risk of contamination by microorganisms. Bone morphogenetic protein is vulnerable to chemicals, but shows resistance to thermal heat to 70 degrees C in a short time. In this experiment, crude human bone morphogenetic protein was extracted from heat-treated bones at 60 degrees C for 10 hours and from nonheated bones. Sodium dodecyl sulfate polyacrylamide gel electrophoresis for these specimens was done. Gelatin capsules containing 5 mg of crude human bone morphogenetic protein extracted from heated and nonheated bones were implanted into thigh muscle pouches of five mice. At 20 days after implantation, the heterotopic bone formation was compared by evaluating the radiographic and histologic analyses. The sodium dodecyl sulfate polyacrylamide gel electrophoresis pattern of the human bone morphogenetic proteins showed five main bands (16, 22, 28, 35, and 67 kDa) that were almost identical. Heterotopic bone formation observed on the radiograph was induced by crude human bone morphogenetic protein from heated bones in a manner similar to that used for nonheated bones. The results from this study show that heat-treated bone preserves osteoinduction.     

Local tumor control with salvage cryotherapy for locally recurrent prostate cancer after external beam radiotherapy

PURPOSE: We identified variables associated with a positive prostate biopsy after salvage cryotherapy in patients in whom initial external beam radiotherapy for prostate cancer failed to improve our cryotherapy technique, optimize local control and improve our patient selection criteria for salvage cryotherapy. MATERIALS AND METHODS: Between July 1992 and January 1995, 145 patients underwent salvage cryotherapy. Post-cryotherapy sextant prostate biopsies were performed in 107 cases. We evaluated certain variables on univariate and multivariate analysis as predictors of a positive biopsy after cryotherapy, including the type of previous therapy, tumor stage and grade at initial diagnosis, prostate volume, pre-cryotherapy prostate specific antigen (PSA), number of positive biopsy cores before cryotherapy, PSA nadir after cryotherapy, stage and grade of local recurrence, number of cryoprobes, number of freeze-thaw cycles and use of a urethral warming catheter during cryotherapy. RESULTS: Biopsies were positive in 23 cases (21%) after salvage cryotherapy. Variables associated with a positive biopsy on univariate analysis were initial stage, precryotherapy PSA, PSA nadir after cryotherapy, number of cryoprobes, number of freeze-thaw cycles and a history of chemotherapy (p = 0.005, 0.027, 0.001, 0.009, 0.018 and 0.008, respectively). Variables that remained associated with a positive biopsy on multivariate analysis were the number of probes used and post-cryotherapy PSA nadir (p = 0.013 and 0.019, respectively). CONCLUSIONS: Patients with initial clinical stage T1-2N0M0 disease and PSA no more than 10 ng./ml. have a higher rate of negative biopsies after salvage cryotherapy. Therefore, they are better candidates for salvage cryotherapy for locally recurrent prostate adenocarcinoma after external beam radiotherapy. To optimize the potential for local control the technique of salvage cryotherapy should include 2 freeze-thaw cycles and a minimum of 5 cryoprobes. Detectable PSA after salvage cryotherapy is a strong predictor of local failure.     

Localized bladder cancer

Opinion statement Transitional cell carcinoma (TCC) of the bladder makes up 90% of bladder cancers. The approach to the management of localized TCC includes accurate clinical and histologic diagnosis and staging with pathologic material obtained through endoscopy. Once the diagnosis of superficial TCC has been established, histologically based prognostic factors guide which therapy or combination of therapies is indicated in the management of individual patients. Surgery alone (transurethral resection) is appropriate initial therapy for noninvasive papillary TCC. For lamina propria invasive tumors and carcinoma in situ, intravesical immunotherapy with bacille Calmette-Guérin (BCG) is often the first line of treatment to decrease tumor recurrence and to possibly decrease progression and improve survival. Intravesical chemotherapy and interferon are alternative therapies that can also decrease recurrence rates. For BCG-refractory TCC, durable response rates with alternative intravesical therapies are low. For superficial TCC that is refractory to endoscopic procedures and intravesical agents or for disease progression, radical cystectomy with neobladder formation or other forms of urinary diversion is the treatment of choice.     

The Presence of CA19-9 in Serum and Saliva from Lewis Blood-group Negative Cancer Patients

Eighteen cancer patients showed high levels of CA19-9 in sera, even though the blood-group phenotypes of their red blood cells were Le(a — b —). Seven of these patients (group I) were determined as Le(a — b —) from both red blood cells and saliva consistently, whereas eleven other patients (group II) secreted either Le^a or Le^b antigen in saliva and showed the expression of incompatible Lewis blood-group antigens. GDP-fucose: N -acetyl-glucosaminide a (1 × 4)-L-fucosyltransferase was demonstrated to be present in salivas from both group I and group II. These results suggest that a cancer-associated alteration of Lewis blood-group antigen expression occurs in cancer patients.     

A novel Src kinase inhibitor, M475271, inhibits VEGF-induced human umbilical vein endothelial cell proliferation and migration

Vascular endothelial growth factor (VEGF) was reported to be a potent proangiogenic factor that plays a pivotal role in both physiological and pathological angiogenesis. M475271, 4-quinazolinamine, N-(2-chloro-5-methoxyphenyl)-6-methoxy-7-[(1-methyl-4-piperidinyl) methoxy]-(9Cl), is a new anilinoquinazoline derivative that showed selective inhibition of Src kinase activity and tumor growth in vivo. Here, we examined the effect of M475271 on VEGF-induced human umbilical vein endothelial cell (HUVEC) proliferation and migration and their intracellular mechanisms. Our findings showed that M475271 pretreatment resulted in a significant inhibition of VEGF-induced HUVEC proliferation, [(3)H]thymidine incorporation, and migration. M475271 inhibited VEGF-induced Flk-1 and Src phosphorylation and their association. Confocal laser microscopic examination confirmed the inhibitory effect of M475271 on VEGF-induced Flk-1/Src association. M475271 inhibited VEGF-induced extracellular signal-regulated kinase1/2 (ERK1/2) and p38 but not Akt activation in a concentration-dependent manner. M475271, PI3-K inhibitor, and p38 inhibitor inhibited VEGF-induced HUVEC proliferation and migration. However, a MEK1/2 inhibitor inhibited VEGF-induced proliferation but not migration. These findings suggest that M475271 attenuates VEGF-induced HUVEC proliferation and migration through the inhibition of signaling pathways involving Src, ERK1/2, and/or p38. Taken together, these data indicate that M475271 may be a useful candidate for inhibition of endothelial cell proliferation and migration relevant to angiogenesis.     

Exercise training enhances tumor necrosis factor-alpha-induced expressions of anti-apoptotic genes without alterations in caspase-3 activity in rat epididymal adipocytes

The effect of exercise training on tumor necrosis factor-alpha (TNF-alpha) signaling was investigated in rat epididymal adipocytes. Incubation of isolated adipocytes with TNF-alpha (20 ng/ml) for 5 h enhanced the expression of the inhibitor apoptosis protein 2 (IAP2) gene without any enhancement of caspase-3 activity in both the sedentary control (C) and exercise-trained (TR) groups. However, the ability of TNF-alpha to enhance IAP2 gene expression was significantly greater in TR than in C rats. The basal expression of the IkappaB kinase beta (IKK beta) gene and that of the BCL-x(L) gene were also higher in TR than in C rats. Mn-superoxidedismutase contents in adipocytes were higher in TR than in C rats. Moreover, no apoptotic nucleuses of adipocytes in response to acute exercise were observed in either group at least up to 5 h after exercise. Exercise training also enhanced the inhibitory effect of TNF-alpha on the gene expression of the fatty acid synthase (FAS), a lipogenic enzyme, suggesting that fatty acid synthesis may be reduced. Thus, exercise training enhanced TNF-alpha signaling directed toward the expressions of survival signals and the suppression of FAS gene expression.     

A critical increase in right-to-left shunt after acute myocardial infarction in a 68-year-old male with tetralogy of Fallot

A male patient with tetralogy of Fallot accompanied by aortic regurgitation had maintained sufficient exercise capacity for a number of decades with the status of acyanotic tetralogy of Fallot. When he was 67 years old, he suffered a posterior wall acute myocardial infarction and direct percutaneous coronary angioplasty successfully revascularised the target lesion in the left circumflex artery. However, a few months after the onset of the myocardial infarction, his shortness of breath became clinically significant and was associated with increased right-to-left shunt and increased right ventricular end-diastolic pressure, as well as hypoxia. At 68 years old, therefore, total corrective repair of the tetralogy with replacement of the aortic and pulmonary valves was performed. The patient was asymptomatic after the successful operation. This report suggests that coronary artery disease can be one of the potential factors in inducing critical hemodynamic changes in aging patients with congenital heart disease, especially those who have a shunt between the right and left chambers. The unique clinical course is described with some discussion of the repair of tetralogy in adults.     

Suppression of visually and memory-guided saccades induced by electrical stimulation of the monkey frontal eye field. II. Suppression of bilateral saccades

To understand the neural mechanism of fixation, we investigated effects of electrical stimulation of the frontal eye field (FEF) and its vicinity on visually guided (Vsacs) and memory-guided saccades (Msacs) in trained monkeys and found that there were two types of suppression induced by the electrical stimulation: suppression of ipsilateral saccades and suppression of bilateral saccades. In this report, we characterized the properties of the suppression of bilateral Vsacs and Msacs. Stimulation of the bilateral suppression sites suppressed the initiation of both Vsacs and Msacs in all directions during and approximately 50 ms after stimulation but did not affect the vector of these saccades. The suppression was stronger for ipsiversive larger saccades and contraversive smaller saccades, and saccades with initial eye positions shifted more in the saccadic direction. The most effective stimulation timing for the suppression of ipsilateral and contralateral Vsacs was approximately 40-50 ms before saccade onset, indicating that the suppression occurred most likely in the superior colliculus and/or the paramedian pontine reticular formation. Suppression sites of bilateral saccades were located in the prearcuate gyrus facing the inferior arcuate sulcus where stimulation induced suppression at < or =40 microA but usually did not evoke any saccades at 80 microA and were different from those of ipsilateral saccades where stimulation evoked saccades at < or =50 microA. The bilateral suppression sites contained fixation neurons. The results suggest that fixation neurons in the bilateral suppression area of the FEF may play roles in maintaining fixation by suppressing saccades in all directions.     

BMK1 is activated in glomeruli of diabetic rats and in mesangial cells by high glucose conditions

BACKGROUND: High glucose causes renal cell injury through various signal transduction pathways, including mitogen-activated protein (MAP) kinases cascades. Big MAP kinase 1 (BMK1), also known as extracellular signal-regulated kinase 5 (ERK5), is a recently identified MAP kinase family member and was reported to be sensitive to osmotic and oxidative stress. However, the role of BMK1 in diabetic nephropathy has not been elucidated yet. METHODS: We investigated whether BMK1 is activated in the glomeruli of Otsuka Long Evans Tokushima Fatty (OLETF) rats, a model of type 2 diabetes mellitus in comparison with the control Long Evans Tokushima Otsuka (LETO) rats. We also examined the effect of high glucose on BMK1 activity in cultured rat mesangial cells. RESULTS: BMK1 and ERK1/2 but not p38 were activated in the glomeruli of OLETF rats, which showed diabetic nephropathy at 52 weeks of age. High glucose, in addition to a high concentration of raffinose, caused rapid and significant activation of BMK1 in rat mesangial cells. MAP kinase/ERK kinase (MEK) inhibitors, U0126 and PD98059, both inhibited BMK1 activation by high glucose in a concentration-dependent manner. Protein kinase C (PKC) inhibition by GF109203X and PKC down-regulation with long-time phorbol myristate acetate (PMA) treatment both inhibited BMK1 and Src kinase activation. Src kinase inhibitors, herbimycin A and PP2, also inhibited high glucose-induced BMK1 activation. PKC inhibitors, Src inhibitors and MEK inhibitors, all inhibited cell proliferation by high glucose. Finally, transfection of dominant-negative MEK5, which is an upstream regulator of BMK1, abolished the BMK1-mediated rat mesangial cell proliferation stimulated by high glucose. CONCLUSION: In the present study, we demonstrated that high glucose activates BMK1 both in vivo and in vitro. It was suggested that high glucose induces PKC- and c-Src-dependent BMK1 activation. It could not be denied that BMK1 activation is induced through an osmotic stress-sensitive mechanism. BMK1-mediated mesangial cell growth may be involved in the pathogenesis of diabetic nephropathy.